In the recombinant production of peptides, especially in the production of pharmaceuticals, the amount of contamination, such as variants of the wanted protein, should be reduced as much as possible both from economical and therapeutical aspects.
In the recombinant production of peptides, variants with an extra sulfur atom in a disulfide bridge sometimes are found, and the present invention relates to this problem.
Human Growth hormone, hGH, is a protein consisting of a single chain of 191 amino acids. The molecule is cross-linked by two disulfide bridges and the monomeric form has a molecular weight of 22 kDa.
hGH preparations have been prepared from human pituitaries, but nowadays the products on the market are produced by recombinant methods, rhGH.
Two types of therapeutically useful recombinant hGH preparations are present on the market: the authentic one, e.g. Genotropin®, Pharmacia & Upjohn AB, and an analogue with an additional methionine residue at the N-terminal end, e.g. Somatonorm®.
hGH is used to stimulate linear growth in patients with hypo pituitary dwarfism or Turner's syndrome but other indications have also been suggested.
A new variant of human growth hormone, hGH, has been found and reference is given to Pavlu et al, 1993, Bioseparation 3, 257-265. This variant has been identified and characterized, see Andersson et al, 1996, Int. J. Peptide, Protein,. Res. 47, 311-321. The variant, which is formed during the expression of hGH in Escherichia coli, is found to be more hydrophobic than rhGH and has been structurally defined as a trisulfide variant of rhGH.
The variant is only formed during synthesis in E Coli and has not been found in hGH preparations from human pituitaries.
This phenomenon of the trifulsides in peptides, produced by recombinant methods, has also been described for recombinant superoxide dismutase (Briggs et al, 1987, Biochem. Biophys. Acta, 537, 100-109) and for a mutein of interleukin, (Breton J et al. J. Chromatogr. A., 1995, 709(1), 135-46).
In WO 94/24127 a method for converting a hydrophobic derivative of a growth hormone into the native form of growth hormone is disclosed. The hydrophobic derivative of the growth hormone comprises an extra sulfur atom. The method is a chemical treatment of the derivative of growth hormone with a mercapto compound. As examples are cystein, gluthatione, 2-mercapto ethanol and dithiothreitol given.
In WO 96/02570 a method is disclosed comprising the chemical treatment with a sulfite compound for the conversion of the derivative of growth hormone into the native form. Mercapto compounds and sulfite compounds are used in the redox-reaction for the conversion of the already formed growth hormone comprising an extra sulfur atom.